Antithrombosis enzyme from the snake venom of agkistrodon acutus

ABSTRACT

This invention features an antithrombosis enzyme extracted and purified from the snake venom of Southern-Anhui Agkistrodon acutus and pharmaceutical uses thereof.

RELATED APPLICATION

This application is a continuation of 09/058,740, filed on Apr. 10,1998, now published U.S. Pat. No. 6,489,451, which claims benefit of60/043,886, filed on Apr. 10, 1997.

FIELD OF THE INVENTION

This invention relates to an antithrombosis enzyme derived from thesnake venom of an acutus species.

BACKGROUND OF THE INVENTION

Anti-thrombus drugs extracted from acutus venom have been reported inthe literature, e.g., “Preparation and Study of Anti-thrombus EnzymesNo. 1, 2, and 3”, Journal of the Medical Univ. of China, 1989.18(special issue); and “Technique for Extracting Definriogenase from thevenom of Agkistrodon acutus,” CN 92102645.5 (CN 1065680.A). Theseanti-thrombus drugs are proteinase components extracted from the snakevenom. They act like thrombase with hemorrhagic side-effect. Inaddition, some of these products are not single component proteinase,but a mixture of different components, which limits the pharmaceuticalapplication of these drugs in human.

Other snake venom derived pharmaceutical products include Ancrod,Trigtamin and Integrilin (see Matsuzaki et al., Biochem. Biophy. Res.Com. 220(2):382-387, 1996; Morita et al., Natural Toxins II, pp187-196,Edited by B. R. Singh and A. T. Tu, Plenum Press, New York, 1996; U.S.Pat. Nos. 5,196,403, 5,242,810, 5,453,370, 4,017,012, 5,344,783,5,686,571, 5,523,292, 5,066,592 and 5,342,830).

SUMMARY OF THE INVENTION

Within the scope of this invention, Applicant has extracted, purifiedand cloned an antithrombosis enzyme (ATE, also called a fibrinolyticenzyme in the provisional application) from the venom of Southern-AnhuiAgkistrodon acutus in China. This enzyme degrades both fibrinogen andfibrin, and inhibits platelet aggregation. It is useful for preventingand treating vaso-occulusive and thromboembolic disorders, including,but not limited to, myocardial infarction, restenosis, peripheralanginaphraxis, angiopathic thrombosis, cerebral thrombosis, ischemiccerebral vascular diseases, unstable angina, acute thrombosis, unstablestenocardia and hemiparalysis caused by cerebral thrombosis.

The present invention provides methods and compositions for preventingor treating diseases and processes mediated (caused or aggravated) byundesired and/or uncontrolled thrombosis by administering to a human oranimal a composition containing or capable of expressing theantithrombosis enzyme in a dosage sufficient to prevent, reduce,eliminate or inhibit thrombosis. The antithrombosis enzyme may besubstantially purified or in a crude extract. The antithrombosis enzymemay be produced from snake venom, chemically synthesized or expressedfrom a recombinant vector. It may also be combined with apharmaceutically acceptable excipient or carrier, and optionallysustained-release compounds or compositions, such as biodegradablepolymers, to form therapeutic compositions.

The present invention is particularly useful for treating or preventingacute and recurrent cerebral thrombosis, myocardial infarction,restenosis, peripheral anginaphraxis, angiopathic thrombosis, ischemiccerebral vascular thrombosis, unstable angina, unstable stenocardia, andthromboangitis obliterans. Administration of the antithrombosisi enzymecan prevent blood clot formation and reduce, diminish or dissolve bloodclot. The antithrombosis enzyme may also be used in combination withother compositions and procedures for the treatment of thrombosis. Forexample, it may be used in combination with a thrombolytic agent knownin the art, which includes, but is not limited to, tissue plasminogenactivator purified from natural sources, recombinant tissue plasminogenactivator, streptokinase, urokinase, prourokinase, anisolatedstreptokinase plasminogen activator complex (ASPAC), animal salivarygland plasminogen activators and known, biologically active derivativesof any of the above. In these combination compositions, theantithrombosis enzyme and other thrombolytic agent work in acomplementary fashion to dissolve blood clots, resulting in decreasedreperfusion times and increased reocclusion times in patients treatedwith them. The use of the antithrombosis enzyme in the compositions ofthis invention advantageously allows the administration of athrombolytic reagent in dosages previously considered too low to resultin thrombolytic effects if given alone. This avoids some of theundesirable side effects associated with the use of thrombolytic agents,such as bleeding complications. The compositions of this invention mayalso be used before, concurrent with, or after angioplastic orfibrolytic treatment to prevent or treat restenosis.

Thus, in a first aspect, this invention features an isolated, purifiedor recombinant antithrombosisi enzyme which has (i) a molecular weightof between about 28 kD and about 32 kD when analyzed by polyacrylamidegel electrophoresis, (ii) an aspartic acid content of between about 2%and about 5%, and (iii) a glutamic acid content of between about 2% andabout 5%. This enzyme has the ability to hydrolyze fibrin, dissolvethrombus, inhibit platelet aggregation, and inhibit the formation ofthrombus.

In a preferred embodiment, the enzyme has fibrinolytic activity of noless than one fibrinolytic activity unit per mg protein. In anotherpreferred embodiment, the enzyme has fibrinolytic activity of betweenabout one and about three fibrinolytic activity units per mg protein.This enzyme specifically hydrolyzes the A (α) chain of fibrinogen. Thisenzyme completely or almost completely inhibits human plateletaggregation induced by agonists such as ADP, Epinephrine, Thrombin andcollagen. This enzyme has no detectable hydrolysis effect on casein. Theenzyme dissolves arterial and venous thrombus in a mammal, preventthrombosis, reduce blood viscosity, and improve microcirculation. At thesame time, this enzyme has minimum effect on the thromosystem, resultingin little possibility of hemorrhage. This enzyme is different fromrelated enzymes from other Acutus species (e.g., IX/X binding proteins,Matsuzaki et al., Biochem. Biophy. Res. Com. 220(2):382-387, 1996;Morita et al., Natural Toxins II, pp187-196, Edited by B. R. Singh andA. T. Tu, Plenum Press, New York, 1996) in that this enzyme has bothfibrinolytic activity and antiplatelet aggregation activity, and lesshemorrhagic activity.

In other preferred embodiments, this enzyme is purified fromSouthern-Anhui Aqkistrodon acutus. The enzyme is a heterodimer of Achain and B chain each with a molecular weight of about 14 KD to about16 KD. The A chain has at its amino end the following sequence:Asp-Cys-Ser-Ser-Asp-Trp-Ser-Ser-Tyr-Glu-Gly-His-Cys-Tyr-Lys-Val-Phe-Lys-Gln-Ser-Lys-Thr-Trp-Thr-Asp-Ala-Glu-Ser-Phe-,and the B chain has at its amino end the following sequence:Asp-Cys-Pro-Ser-Glu-Trp-Ser-Ser-Tyr-Glu-Gly-Phe-Cys-Tyr-Lys-Pro-Phe-.Preferrably, the A chain and the B chain are linked by one or moredisulfide bond.

In other preferred embodiments, this antithrombosis enzyme contains Ca⁺⁺and/or has aspartic acid at its amino terminus.

By “isolated” in reference to a polypeptide is meant a polypeptideisolated from a natural source or synthesized. The isolated polypeptidesof the present invention are unique in the sense that they are not foundin a pure or separated state in nature. Use of the term “isolated”indicates that a naturally occurring amino acid sequence has beenremoved from its normal cellular environment. Thus, the sequence may bein a cell-free solution or placed in a different cellular environment.The term does not imply that the sequence is the only amino acid chainpresent, but that it is the predominate sequence present (at least10-20% more than any other sequence) and is essentially free (about90-95% pure at least) of non-amino acid material naturally associatedwith it.

By “enriched” in reference to a polypeptide is meant that the specificamino acid sequence constitutes a significantly higher fraction (2-5fold) of the total of amino acids present a in the cells or solution ofinterest than in normal or diseased cells or in the cells from which thesequence was taken. This could be caused by a person by preferentialreduction in the amount of other amino acids present, or by apreferential increase in the amount of the specific amino acid sequenceof interest, or by a combination of the two. However, it should be notedthat enriched does not imply that there are no other amino acidsequences present, just that the relative amount of the sequence ofinterest has been significantly increased. The term “significantly” hereis used to indicate that the level of increase is useful to the personmaking such an increase, and generally means an increase relative toother amino acids of about at least 2 fold, more preferably at least 5to 10 fold or even more. The term also does not imply that there is noamino acid from other sources. The amino acid from other sources may,for example, comprise amino acid encoded by a yeast or bacterial genome,or a cloning vector such as pUC19. The term is meant to cover only thosesituations in which man has intervened to elevate the proportion of thedesired amino acid.

By “purified” in reference to a polypeptide does not require absolutepurity (such as a homogeneous preparation); instead, it represents anindication that the sequence is relatively purer than in the naturalenvironment (compared to the natural level this level should be at least2-5 fold greater, e.g., in terms of mg/ml). Purification of at least oneorder of magnitude, preferably two or three orders, and more preferablyfour or five orders of magnitude is expressly contemplated. Thesubstance is preferably free of contamination at a functionallysignificant level, for example 90%, 95%, or 99% pure.

By “recombinant” is meant a polypeptide or enzyme produced byrecombinant DNA techniques such that it is distinct from a naturallyoccurring polypeptide either in its location (e.g., present in adifferent cell or tissue than found in nature), purity or structure.Generally, such a recombinant polypeptide will be present in a cell inan amount different from that normally observed in nature. Thisinvention features recombinant ATE and its fragments obtainable usingtechniques known to those skilled in the art, including those describedin McDonnell et al., U.S. patent application Ser. No. 08/223,943 filedApr. 6, 1994, Evans et al., U.S. Pat. No. 5,071,773, and PCTapplication, PCT/US91/00399 filed Jan. 22, 1991 (InternationalPublication No. WO 91/12258), incorporated by reference herein.

In a second aspect, this invention features isolated, purified orrecombinant polypeptide fragments of the A chain and the B chain of theantithrombosis enzyme. Preferably, these fragments contain no less than15, 20, 30 or 40 contiguous amino acid residues from the A or B chain.For example, these fragments may contain no less than 15, 20, 30 or 40contiguous amino acid residues from SEQ ID NO: 2. Such polypeptidefragments can be synthesized chemically or expressed from recombinantvectors. They are useful for generating monoclonal antibodies which bindto both the polypeptide fragments and the intact antithrombosis enzyme(see U.S. Pat. Nos. 5,733,738, 5,015,571, incorporated by referenceherein). Monoclonal antibodies so generated can be attached to solidsupport and used to purify the antithrombosis enzyme from crude venomextract or cell extract by affinity chromatography.

The recombinant polypeptide fragments of the A chain and the B chain canbe expressed from recombinant nucleic acid encoding such polypeptidefragments. For example, polypeptide fragments of the A chain can beexpressed from recombinant nucleic acid containing no less than 45, 60,90 or 120 contiguous nucleotides from SEQ ID NO: 1 or its fullycomplementary strand of the same length and a promoter effective toinitiate transcription of the contiguous nucleotides in a host cell.

In yet another aspect the invention features an isolated, enriched, orpurified antibody (e.g., a monoclonal or polyclonal antibody) havingspecific binding affinity to the antithrombosis enzyme or a fragmentthereof. The antibody contains a sequence of amino acids that is able tospecifically bind to the antithrombosis enzyme. The antibody may beprepared with techniques known to those skilled in the art, including,but not limited to, those disclosed in Niman, PCT applicationPCT/US88/03921 (International Publication No. WO 89/04489), incorporatedby reference herein. By “specific binding affinity” is meant that theantibody will bind to the ATE in a certain detectable amount but willnot bind other polypeptides to the same extent under identicalconditions.

In another aspect the invention features a hybridoma which produces anantibody having specific binding affinity to the antithrombosis enzymeor a fragment thereof. By “hybridoma” is meant an immortalized cell linewhich is capable of secreting an antibody.

In another aspect, the invention features an isolated, purified,enriched or purified recombinant nucleic acid encoding theantithrombosis enzyme, a chain of the enzyme, or fragments of the Achain or B chain. For example, the recombinant nucleic acid contains asequence contiguously encoding SEQ ID NO: 2 and a promoter effective toinitiate transcription of the conding sequence in a host cell. Inparticular, the recombinant nucleic acid contains SEQ ID NO: 1 operablylinked to a promoter.

By “isolated” in reference to nucleic acid is meant DNA or RNA isolatedfrom a natural source or synthesized. The isolated nucleic acid of thepresent invention is unique in the sense that it is not found in a pureor separated state in nature. Use of the term “isolated” indicates thata naturally occurring sequence has been removed from its normal cellularenvironment. Thus, the sequence may be in a cell-free solution or placedin a different cellular environment. The term does not imply that thesequence is the only nucleotide chain present, but does indicate that itis the predominate sequence present (at least 10-20% more than any othernucleotide sequence) and is essentially free (about 90-95% pure atleast) of non-nucleotide material naturally associated with it.Therefore, the term does not encompass an isolated chromosome encodingthe ATE.

By “purified” in reference to nucleic acid does not require absolutepurity (such as a homogeneous preparation); instead, it represents anindication that the sequence is relatively purer than in the naturalenvironment (compared to the natural level this level should be at least2-5 fold greater, e.g., in terms of mg/ml). Individual clones isolatedfrom a cDNA library may be purified to electrophoretic homogeneity. Theclaimed DNA molecules obtained from these clones could be obtaineddirectly from total DNA or from total RNA. The cDNA clones are notnaturally occurring, but rather are preferably obtained via manipulationof a partially purified naturally occurring substance (messenger RNA).The construction of a cDNA library from mRNA involves the creation of asynthetic substance (cDNA) and pure individual cDNA clones can beisolated from the synthetic library by clonal selection of the cellscarrying the cDNA library. Thus, the process which includes theconstruction of a cDNA library from mRNA and isolation of distinct cDNAclones yields an approximately 10⁶-fold purification of the nativemessage. Thus, purification of at least one order of magnitude,preferably two or three orders, and more preferably four or five ordersof magnitude is expressly contemplated.

By “enriched” in reference to nucleic acid is meant that the specificDNA or RNA sequence constitutes a significantly higher fraction (2-5fold) of the total DNA or RNA present in the cells or solution ofinterest than in normal or diseased cells or in the cells from which thesequence was taken. This could be caused by a person by preferentialreduction in the amount of other DNA or RNA present, or by apreferential increase in the amount of the specific DNA or RNA sequence,or by a combination of the two. However, it should be noted thatenriched does not imply that there are no other DNA or RNA sequencespresent, just that the relative amount of the sequence of interest hasbeen significantly increased in a useful manner and preferably separatefrom a sequence library. The term “significantly” here is used toindicate that the level of increase is useful to the person making suchan increase, and generally means an increase relative to other nucleicacids of about at least 2 fold, more preferably at least 5 to 10 fold oreven more. The term also does not imply that there is no DNA or RNA fromother sources. The DNA from other sources may, for example, comprise DNAfrom a yeast or bacterial genome, or a cloning vector such as pUC19.This term distinguishes from naturally occurring events, such as viralinfection, or tumor type growths, in which the level of one mRNA may benaturally increased relative to other species of mRNA. That is, the termis meant to cover only those situations in which a person has intervenedto elevate the proportion of the desired nucleic acid.

By “recombinant” in reference to a nucleic acid is meant the nucleicacid is produced by recombinant DNA techniques such that it is distinctfrom a naturally occurring nucleic acid.

By “comprising” is meant including, but not limited to, whatever followsthe word “comprising”. Thus, use of the term “comprising” indicates thatthe listed elements are required or mandatory, but that other elementsare optional and may or may not be present. By “consisting of” is meantincluding, and limited to, whatever follows the phrase “consisting of”.Thus, the phrase “consisting of” indicates that the listed elements arerequired or mandatory, and that no other elements may be present. By“consisting essentially of” is meant including any elements listed afterthe phrase, and limited to other elements that do not interfere with orcontribute to the activity or action specified in the disclosure for thelisted elements. Thus, the phrase “consisting essentially of” indicatesthat the listed elements are required or mandatory, but that otherelements are optional and may or may not be present depending uponwhether or not they affect the activity or action of the listedelements.

The invention also features a nucleic acid probe for the detection of anucleic acid encoding the antithrombosis enzyme, its A chain or B chain,or fragments thereof. In an example, the nucleic acid probe containsnucleic acid that will hybridize to SEQ ID NO: 1, but not to the nucleicacid sequence of the IX/X-binding protein (Matsuzaki et al., Biochem.Biophy. Res. Com. 220(2):382-387, 1996) under high stringencyhybridization conditions. By “high stringency hybridization conditions”is meant those hybridizing conditions that (1) employ low ionic strengthand high temperature for washing, for example, 0.015 M NaCl/0.0015 Msodium citrate/0.1% SDS at 50° C.; (2) employ during hybridization adenaturing agent such as formamide, for example, 50% (vol/vol) formamidewith 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodiumcitrate at 42° C.; or (3) employ 50% formamide, 5×SSC (0.75 M NaCl,0.075 M Sodium pyrophosphate, 5×Denhardt's solution, sonicated salmonsperm DNA (50 g/ml), 0.1% SDS, and 10% dextran sulfate at 42° C., withwashes at 42° C. in 0.2×SSC and 0.1% SDS. Under stringent hybridizationconditions only highly complementary nucleic acid sequences hybridize.Preferably, such conditions prevent hybridization of nucleic acidshaving 1 or 2 mismatches out of 20 contiguous nucleotides.

In another aspect, the invention describes a recombinant cell or tissuecontaining an exogenous nucleic acid coding for the antithrombosisenzyme, its A chain or B chain, or fragments thereof.

In another aspect, this invention features a pharmaceutical compositionfor reducing or eliminating thrombosis in a human or animal subject.This composition contains a pharmaceutically effective amount of theantithrombosis enzyme and a pharmaceutically acceptable carrier.

By “pharmaceutically effective amount” is meant an amount of apharmaceutical compound or composition having a therapeutically relevanteffect on thrombosis or diseases or pathological conditions related tothrombosis. A therapeutically relevant effect prevents or relieves tosome extent one or more symptoms of thrombosis or diseases orpathological symptoms related to thrombosis in a patient or returns tonormal either partially or completely one or more physiological orbiochemical parameters associated with or causative of thrombosis ordiseases or pathological symptoms related to thrombosis, e.g., reducingor inhibiting thrombosis; curing, reducing, or inhibiting diseases andprocesses that are mediated by thrombosis.

In another aspect, this invention features a method of reducing orinhibiting thrombosis in a human or animal subject by administering tothe subject a pharmaceutically effective amount of the antithrombosisenzyme.

In another aspect, this invention features a method of isolating and/orpurifying the antithrombosis enzyme. In this method, a crude extractcontaining the antithrombosis enzyme, e.g., a crude extract of the snakevenom of Southern-Anhui Agkistrodon acutus, is dissolved in a buffer andbrought into contact with an anion exchange column, the elution samplewith fibrinolytic activity and antiplatelet aggregation activity iscollected, concentrated and separated by gel permeation chromatography,and the elution sample with fibrinolytic activity and antiplateletaggregation activity is collected and desalted. This invention featuresan antithrombosis enzyme isolated by this process.

The present invention also provides recombinant DNA moleculescharacterized by a DNA sequence encoding the antithrombosis enzyme aloneor fused to a DNA sequence which codes for a conventionalanti-thrombosis polypeptide. The synthesis of these DNA molecules may beachieved by methods well known in the art. For example, theserecombinant DNA molecules may be isolated from an Agkistrodon acutusvenom gland cDNA library. The synthesis of cDNA libraries and the choiceof vector into which the cDNA molecules may be cloned are conventionaltechniques. The invention also relates to hosts transformed with theserecombinant DNA molecules, as well as to the recombinant productsexpressed by these hosts. And the present invention relates tochemically synthesized antithrombosis enzyme. Such syntheticpolypeptides may be prepared by conventional chemical synthesistechniques, for example, synthesis on a solid support.

This invention further relates to pharmaceutically acceptablecompositions and combinations, and methods utilizing these natural,recombinant or synthetic antiplatelet polypeptides in the treatmentextracorporeal blood.

By “extracorporeal blood” is meant blood that is removed from a patient,subjected to extracorporeal treatment, and returned to the patient inprocesses such as dialysis, blood filtration or blood bypass duringsurgery. The term also includes blood products which are storedextracorporeally for eventual administration to a patient. Such productsinclude whole blood, platelet concentrates and other blood fractions inwhich inhibition of platelet aggregation and platelet release isdesired.

Other features and advantages of the invention will be apparent from thefollowing drawing and detailed description of the invention and from theclaims.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows the putative cDNA sequence and amino acid sequence of theantithrombosis enzyme, B chain. A segment of about 10-20 nucleotides inthe cDNA clone is being sequenced. A leader peptide—M G R F I F V S F GL L V V F L S L S G T A A—is cleaved before the assembly of A chain andB chain to form the antithrombosis enzyme.

DETAILED DESCRIPTION OF THE INVENTION

The present invention includes compositions and methods for thetreatment of diseases and pathological conditions. These diseases orpathological conditions are mediated by or associated with thrombosis.The compositions of this invention contain an antithrombosis enzyme,which can be isolated from the venom of Southern-Anhui Agkistrodonacutus, or synthesized by chemical or biological methods (e.g., cellculture, recombinant gene expression, or peptide synthesis). Recombinanttechniques include gene amplification from DNA sources using thepolymerase chain reaction (PCR), RNA polymerase based amplification, andgene amplification from RNA sources using reverse transcriptase/PCR.

Purification of the Antithrombosis enzyme from Snake Venom

The antithrombosis enzyme of this invention was extracted and purifiedfrom the venom of Southern-Anhui Agkistrodon acutus in China:

(1) Dissolve the crude snake venom in buffer, proceed with centrifugalprecipitation and chromatographic separation through an anion exchangecolumn, and collect roughly-isolated fibrinolytic component by elution.The elution is conducted with both a pH linear gradient and a saltlinear gradient. The pH linear gradient is from pH 7.5-8.5 to pH6.0-7.0; and the salt linear gradient is from 0 to 0.5 M. The elutionspeed is 25-80 ml/hr.

(2) Concentrate this fibrinolytic component and proceed with gelpermeation chromatography to get the refined antithrombosis enzymesolution.

(3) Desalt the refined antithrombosis enzyme solution and get thepurified antithrombosis enzyme solution.

The above procedure was conducted at about 4-10° C.

The buffer used to dissolve the crude snake venom and theroughly-isolated elute is a Tris-Hcl buffer (e.g. 0.02 M, pH 7.5-8.5N-(tris-hydroxymethyl)-amino methane-Hydrochloric acid), or a phosphatebuffer (e.g. 0.02 M, pH 7.5-8.5 Na₂HPO₄-NaH₂PO₄).

The anion exchange column contains weak acidic anion exchanger, e.g.,DEAE Sephadex A50, or DEAE Sepharose Fast Flow.

The gel permeation chromatography uses a gel for separating substancesin the range between about 5 kD and 300 kD, e.g., Sephacryl S-100HR,S-200HR, S-300HR, or Sephadex G75. The buffer used in the gel permeationchromatography is a 0.05-0.30 M sodium chloride (NaCl) solution.

The fibrinolytic component was concentrated by ultrafiltration,blow-drying with cold air, or freeze-drying. The desalting was conductedagainst distilled water for 1-6 hrs.

The above method for isolating and purifying the antithrombosis enzymefrom the venom of Agkistrodon acutus is simple, provides high-purityproduct and shortens the time by processing the pH linear gradientelution and the salt linear gradient elution simultaneously. Theshortened purification cycle increases the efficiency of industrialproduction.

The purification methods of this invention are also useful to isolateproteolytic fragments of the antithrombosis enzyme which retain thebiological activity of the intact polypeptide. Such proteolyticfragments may occur naturally or artifactually as a result of thepurification process.

The purification procedure is further described by the following threeexamples:

EXAMPLE 1

(1) Dissolve 3 g Southern-Anhui Agkistrodon actus venom in 20 ml 0.02 MpH 8.0 Tris-Hcl buffer. Centrifuge 15 min at 3000 rpm. Collect thesupernatant. Resuspend the pellet in 5 ml above buffer and agitate well.Repeat the centrifugation once. Collect the supernatant with the earliercollection. Store at 4° C.

(2) Process the DEAE Sephadex A-50 anion exchanger with alkaline-acidand wash with distilled water to neutrality. Then wash with 0.02 M pH8.0 Tris-HCl buffer till the eluate's pH reaches 8.0. Keep inboiling-water bath for 2 hrs to remove air bubbles and ensure swelling.

(3) Pack the column with the processed gel from Step (2) when it iscooled to ambient temperature. The column volume is 3×80 cm³. Afterequilibration, load the supernatant sample from Step (1) and elute withlinear gradient buffer. The buffer in the stock bottle is 0.02 MTris-Hcl, pH 6.3, containing 0.5 M NaCl. The buffer in the gradientbottle is 0.02 M Tris-HCl buffer, pH 8.0. The flow rate is 42 ml/hr.Examine the eluate under UV 280 nm. Collect the eluate 48 hours afterthe sample is loaded by automatic fraction collector at the rate of 7 mlper tube and 6 tubes per hour. 12 elution peaks are obtained, amongwhich the 6th is the roughly-isolated fibrinolytic component. It isapproximately a 120 ml solution containing 300 mg enzyme.

(4) Put the above fibrinolytic component in a dialysis bag. Blow the bagwith cold air till its volume is decreased to about 15 ml. Store at 4°C.

(5) Use Sephacryl S0200HR as gel chromatographic medium to pack the gelchromatographic column. The column volume is 2.7×70 cm³. Wash with 0.5 MNaCl solution for 1 hr at the flow rate of 99 ml/hr, then equilibratewith 0.15 M NaCl solution at the flow rate of 18 ml/min. The secondelution peak is the refined antithrombosis enzyme solution whichcontains 170 mg enzyme in 70 ml of solution.

(6) Put the above refined antithrombosis enzyme into a dialysis bag.Dialyze for 3 hrs against distilled water and get 170 mg purifiedantithrombosis enzyme.

The purified enzyme was tested to have 1 fibrinolytic activity unit permg protein. It was tested to show an isoelectric point of pH 5.8 and MWof about 30 kD. No hemorrhagic side effect was detected with the enzyme.

EXAMPLE 2

(1) Dissolve 4.5 g Southern-Anhui Agkistrodon acutus venom in 0.02 M, pH7.8 phosphate buffer. The rest is the same as Step (1) of Example 1.Combine the supernatant and store at 4° C.

(2) Same as Step (2) of Example 1.

(3) Pack the column with the processed gel by the above step. Increasethe column volume to 1600 ml (4×100 cm³). Keep the other conditions, 460mg of enzyme was obtained after the chromatography.

(4) Put the roughly-isolated antithrombosis enzyme in a dialysis bag.Blow the bag with cold air till the volume is decreased to about 20 ml.

(5) Use Sephacryl S-300 HR as gel chromatographic medium to pack thechromatographic column. The column volume is 3.1×70 cm³. Wash 1 hr with0.5 M NaCl at the flow rate of 99 ml/hr and equilibrate with 0.2 M NaCl.Load the concentrated sample onto the column. Elute with 0.20 M NaClsolution at the flow rate of 24 ml/min. The second peak is the refinedantithrombosis enzyme solution. Desalt the solution and get 280 mgpurified enzyme in 100 ml solution.

EXAMPLE 3

(1) Dissolve 3 g Agkistrodon acutus venom as described in Step (1) ofExample 1.

(2) Use DEAE Sepharose Fast Flow as the anion exchanger. Process it andpack the column as described in Step (2) of Example 1. The column volumeis 3×80 cm³. Rinse the column with 0.02 M pH 8.0 Tris-HCl buffer at theflow rate of 1.5 ml/min till equilibration. Load the sample and elutewith a linear gradient. The elution buffer is the same at that in Step(3) of Example 1. Adjust the flow rate to 80 ml/hr. 285 mg of enzyme wascollected.

(3) The following procedure is the same as that of Example 1. In the end160 mg refined antithrombosis enzyme in 60 ml solution was obtainedwhich was shown to have fibrinolytic activity and no hemorrhagicside-effect.

Crystallization of the Antithrombosis Enzyme

The antithrombosis enzyme was obtained as described above (with a purityof 99% or higher) and further desulted. Crystals of the antithrombosisenzyme were grown by hanging-drop vapour diffusion technique usingtissue culture plates and silicomised glass coverslips. At roomtemperature (about 20° C.), 3 μl of protein solution (30 mg/ml, indistilled water) was mixed with an equal volume of precipitant solution(0.1 M Mes buffer, pH 6.5, containing 16% PEG4000 (w/v) and 0.1 MCaAc2). The solution was then equilibrated against 400 μl of precipitantsolution containing 20% PEG 4000 (w/v). Crystals were collected from thesolution in about one week. These crystals are useful for decipheringthe tertiary structure and function center of the enzyme by X-raycrystallography and other techniques.

Characterization of the Antithrombosis Enzyme

The antithrombosis enzyme purified from the snake venom ofSouthern-Anhui Agkistrodon acutus contains Ca⁺⁺. Applicant studied thepurity, fibrinolytic characteristic, antiplatelet aggregation activity,amino acid structure, proteolytic activity and hemorrhagic activity ofthe antithrombosis enzyme.

The activities of the antithrombosis enzyme of the present invention maybe assayed in vitro using any conventional technique. Preferably, theanti-thrombin assay involves direct determination of thethrombin-inhibitory activity of the molecule. Such techniques measurethe inhibition of thrombin-catalyzed cleavage of colorimetric substratesor the increase in thrombin times or increase in activated partial itthromboplastin times of human plasma. Alternatively, the assay employedmay use purified thrombin and fibrinogen to measure the inhibition ofrelease of fibrinopeptides.

The antiplatelet activity can be measured through a change in the degreeof aggregation of platelets or a change in the release of a plateletsecretory component in the presence of platelet activator. The formermay be measured in an aggregometer. The latter may be measured using RIAor ELISA techniques specific for the secreted component.

Purity

Polyacrylamide gel electrophoresis of the enzyme as purified above undernonreducing condition showed one band at about 28-30 kD and the purityto be more than 90%.

Components

SDS-polyacrylamide gel electrophoresis was carried out according to themethod of Laemmli (Nature 227:680-685,1970). Standards used formolecular weight determination were obtained from Sigma (bovine serumalbumin −66 kD, ovalbumin −45 kD, glyceraldehyde-3-phosphatedehydrogenase −36 kD, carbonic anhydrase −29 kD, trypsinogen −24 kD andlactalbumin −14 kD). Staining was done with Coomassie brilliant blueR250.

Two bands were present on the SDS-PAGE gel. Thus the enzyme was isolatedinto two chains, A chain and B chain, each with a molecular weight ofabout 14 kD to about 16 kD on SDS-PAGE.

Fibrinolytic Activity

Fibrinolytic activity was measured with the fibrin-plate-clearance assay(Bajwa et al. Toxicon 18:285-290, 1980). The positive control and thenegative control were plasmin (Sigma) and 0.9% sodium chloride,respectively. The experiment showed that the enzyme has highfibrinolytic activity in comparison to the control and the size of lysisarea on the plate was proportional to the enzyme dosage. The enzyme wasmeasured to hydrolyze fibrin at the level of about one to about threefibrinolytic activity units per mg of enzyme.

Fibrinogen Cleavage Sites

Fibrinogen was incubated with the antithrombosis enzyme to determine theinitial cleavage sites. Fibrinogen (30 nmol) was dissolved in 2 ml of0.05 M Tris buffer, pH 8.0. 10 μl of the antithrombosis enzyme (0.6nmol) was added to this solution. The solution was incubated at 37° C.for 1 hr. The reaction was stopped by the addition of 1 ml of 250 mMEDTA. This incubation time was employed as being optional for theaccumulation of early degradation products.

The solution was then brought to 8 M Urea. Degraded fibrinogen fragmentswere reduced by the addition of dithiothreitol in a 10-fold molar excessover fibrinogen.

The sample was added to a C₁₈ reversed phase chromatography column forseparation. Five fragments were collected and freeze-dried. Theamino-terminal amino acid sequences (1-10 residues) were determined. Onefibrinogen cleavage site was found to be Met 235-Pro236 of the α chainof human fibrinogen. Other cleavage sites are still being studied.

Antiplatelet Aggregation Activity

ADP, collagen, epinephrine bitartrate and thrombin were obtained fromSigma Chemical Co. (St Louis, Mo.). Venous blood was collected fromhealthy human donors who were drug-free and aspirin-free for at least 2weeks before blood collection. Blood was collected into ciratedVacutainer tubes and centrifuged for 15 minutes at 150 g at roomtemperature. The platelet-rich plasma (PRP) was removed. The remainingblood was centrifuged for 15 minutes at 1500 g at room temperature, andthe platelet-poor plasma (PPP) was removed.

Samples were assayed on a TYSM-91 Platelet Aggregometer with PPP as theblank (100% transmittance). PRP (200 μl, 5×10⁸ platelets/ml) was addedto each micro test tube, and transmittance was set at 0%.

A stimulator of platelet aggregation such as ADP (200 μl, 10 μmol/l),collagen, epinephrine bitartrate or thrombin was added to a tube, andthe aggregation profiles were plotted (percent transmittance versustime). The antithrombosis enzyme (20 μl) of varying concentrations wasadded to the test tube before the addition of the platelet agonist.Antiplatelet aggregation activity is expressed as percent inhibition ofagonist-induced platelet aggregation.

It was shown that the antithrombosis enzyme inhibits human plateletaggregation induced by ADP, Epinephrine, thrombin and collagen.

Primary Structure

The amino acid sequences of the A chain and the N-terminal of the Bchain have been determined:

     A: Asp-Cys-Ser-Ser-Asp-Trp-Ser-Ser-Tyr-Glu-Gly-His-Cys-Tyr-Lys-Val-Phe-Lys-Gln-Ser-Lys-Thr-Trp-Thr-Asp-Ala-Glu-Ser-Phe-Cys-Thr-Lys-Gln-Val-Asn-Gly-Gly-His-Leu-Val-Ser-Ile-Glu-Ser-Ser-Gly-Glu-Ala-Asp-Phe-Val-Gly-Gln-Leu-Ile-Ala-Gln-Lys-Ile-LyS-Ser-Ala-LyS-Ile-His-Val-Trp-Ile-Gly-Leu-Arg-Ala-Gln-Asn-Lys-Glu-Lys-Gln-Cys-Ser-Ile-Glu-Trp-Ser-Asp-Gly-Ser-Ser-Ile-Ser-Lys-Glu-Asn-Trp-Ile-Glu-Glu-Glu-Ser-Lys-Lys-Cys-Leu-Gly-Val-His-Ile-Glu-Thr-Gly-Phe-His-Lys-Trp-Glu-Asn-Phe-Tyr-Cys-Glu-Gln-Gln-Asp-Pro-Phe-Val-Cys-Glu- Ala     B: N-Asp-Cys-Pro-Ser-Glu-Trp-Ser-Ser-Tyr-Glu-Gly-Phe-Cys-Tyr-Lys-Pro- Phe-

The amino acid sequence of the A chain was determined by a combinationof deduction from the cDNA sequence and amino acid sequencing of theN-terminal of purified A chain.

FIG. 1 shows the putative amino acid sequence of the entire B chain,including a leader peptide, i.e., M G R F I F V S F G L L V V F L S L SG T A A, which is cleaved before the assembly of A chain and B chain toform the antithrombosis enzyme.

Proteolytic Activity

The proteolytic activity of the antithrombosis enzyme was assessed byazocasein hydrolysis. Azocasein was synthesized from casein anddiazotised sulfanilamide. The resulting azocasein solution in 1% sodiumbicarbonate had a concentration of 42 mg/ml. 1 ml of the azocaseinsolution was added to a tube. A solution of the antithrombosis enzymewas added, and the tube was incubated at 37° C. for 30 minutes. Theperchloric acid was added into the tube, and the resulting precipitatewas removed by centrifugation. Hydrolysis of azocasein was measured byincreased absorbance at 390 nm of the supernatant. No proteolyticactivity was detected in this assay.

Hemorrhagic Activity

The antithrombosis enzyme (100-200 μg) was injected subdermally underthe clean shaven backs of white mice. After 24 hours, the mice weresacrificed and their skin observed for hemorrhage. No hemorrhage on theskin was observed after the administration of the antithrombosis enzyme.

Pharmacology of the Antithrombosis Enzyme in Animal Studies Effect onThrombosis

The antithrombosis enzyme was administered to rabbits intravenously.Thrombosis was determined before the administration and after theadministration at 0.5, 1.0, 2.0, and 3.0 hours by the Chandler method.The enzyme showed anti-thrombosis activity at 0.5 hr followingadministration at 0.005 u/kg and this activity was increasedsignificantly at 1.0 hr and at 0.01 u/kg.

Effect on Arteria Thrombus and Venous Thrombus

Rats were anesthetized and their abdominal aorta and abdominal vein wereexposed after surgery. Threads were passed through the abdominal aortaand abdominal vein. After two hours, the antithrombosis enzyme wasinjected from the femoral vein. Four hours later, the threads were takenout to observe the arteria thrombus and the venous thrombus on thethread. The enzyme was shown to dissolve the arteria thrombus and thevenous thrombus starting at 0.025 u/kg and effectively at 0.05 u/kg.

Effect on Pulmonary Thrombus

Pulmonary thrombus was detected and measured with ¹²⁵I labeledfibrinogen. The radiation strength in the blood was determined withisotope counting method after the administration of the enzymeintravenously. The enzyme was shown to dissolve pulmonary thrombus inrats effectively at 0.005, 0.01, and 0.02 u/kg.

Effect on Fibrinogen

The enzyme was administered to rabbits intravenously. The content offibrinogen in the plasma was determined at different times afteradministration. The enzyme was shown to reduce the fibrinogen content inthe blood at 0.0025 u/kg, 0.005 u/kg, and 0.01 u/kg.

Effect on Platelet Aggregation

Dogs were anesthetized and placed on a respiratory pump with the strokevolume adjusted according to the dog's weight. The respirator dials wereset to 20 beats per minute and 50% respiration. Both femoral arterieswere cannulated, one was attached to a transducer by which the heartbeat and blood pressure were monitored on a chart recorder throughoutthe test, and the other was, used for repeated blooding sampling. Theformal vein was also cannulated to administer antithrombosis enzyme orvehicle and for the maintenance of anesthesia as needed.

Animals were dosed intravenously with antithrombosis enzyme at differentdosage levels. Bleeding time and platelet aggregation were determinedbefore and at different intervals after the administration of theantithrombosis enzyme. Aspirine (35 mg/kg) was used as the positivecontrol and 0.9% sodium cloride was used as the negative control.

It was shown that the antithrombosis enzyme prolongs the blooding timeand inhibits platelet aggregation.

Effect on ELT

The enzyme was administered to rabbits intravenously. ELT in the bloodof the rabbits was determined at different times after administration.The enzyme was shown to shorten ELT effectively starting at about 120minutes from administration.

Effect on the Content of FDP in the Blood

The enzyme was administered to rabbits intravenously. The content of FDPin the blood of the rabbits was determined at different times afteradministration. The enzyme was shown to increase the content of FDP inthe blood significantly in a dosage dependent manner.

Effect on The Whole Blood Clotting Time

The enzyme was administered to rabbits intravenously. The whole bloodclotting time (CT) was determined. Two hours following theadministration, the enzyme showed anti-clotting activity.

Effect on the Kaolin Partial Thromboplastin Time

The enzyme was administered to rabbits intravenously. The KPTT wasdetermined. 240 minutes following the administration, the enzyme wasshown to prolong the KPTT significantly.

Effect on Thrombin Time

The enzyme was administered to rabbits intravenously. The TT wasdetermined at the different time after administration. 240 minutesfollowing the administration, the enzyme was shown to prolong the TTsignificantly.

Effect on Blood Viscosity

The enzyme was administered to rabbits intravenously. The bloodviscosity was determined at different times after administration.120-240 minutes following the administration, the enzyme was shown toreduce the blood viscosity significantly.

General Pharmacology

Dogs were anesthetized with pentobarbital sodium (30 mg/kg). Theantithrombosis enzyme at dosages in the range of 0.0015-0.06 u/kg wasadministrated intravenously. The left arteria carotis was isolated. Theaortic systolic pressure, diastolic pressure, mean pressure and heartrate were recorded directly by TP-200T pressure transducer and RM-6000multi-channel physiological recorder. At the same time, the respiratorycurve and the electrocardiogram were recorded. The enzyme did not showsignificant effects on the above parameters.

When the enzyme was administered to mice intravenously from the tail, itdid not affect the behavior of the mice. The enzyme did not activate orrestrain the neural system of the mice.

Pharmacokinetics

The pharmacokinetics of the enzyme in the rats was studied with ¹²⁵Ilabeled antithrombosis enzyme at 0.02, 0.01, and 0.005 u/kg. TheConcentration-Time Curves of the enzyme in the blood were very similarat the three dosages which is in agreement with the Two-Room Model.T_(1/2) is 6 hours. Among organs, the enzyme distributes mainly in thelung and the kidney. The enzyme can infiltrate through theblood-brain-barrier. It is excreted mainly through urine.

Cloning and Expression of the Antithrombosis Enzyme

A wide variety of methods may be used in locating and identifying cDNAsequences corresponding to A chain and B chain of this invention. Thetwo most preferred techniques are the use of oligonucleotide probe basedon the amino acid sequences of A chain and B chain and immunoscreening,which utilizes antibodies against A chain or B chain to detect cloneswhich express cDNA sequences corresponding to the polypeptide.

The immuno screening technique requires that the cDNA library becontained in an expression vector. Such vectors include lambda gt11,lambda gt10 and other expression vectors known in the art. Antibodiesemployed in the immunoscreening technique include antibodies against theintact antithrombosis enzyme of the present invention, antibodiesagainst denatured polypeptide and antibodies against peptide portions ofthe antithrombosis enzyme. Once a cDNA clone has been identified andisolated, it may be removed from the vector and analyzed to determinewhether it contains the entire antithrombosis enzyme coding sequence.Partial cDNAs may themselves be used to reprobe the cDNA library and tolocate full-length cDNAs.

The DNA molecules of this invention may be synthesized fromoligonucleotides by chemical means using an oligonucleotide synthesizer.Such oligonucleotides may be designed based on the disclosed amino acidsequences of the antithrombosis enzyme.

Standard methods may be applied to synthesize a gene encoding theantithrombosis enzyme with knowledge of the enzyme's amino acidsequence. For example, the complete amino acid sequence may be used toconstruct a back-translated gene. A DNA oligomer containing a nucleotidesequence capable of coding for the desired antithrombosis enzyme may besynthesized in a single step. Alternatively, several smalleroligonucleotides coding for portions of the antithrombosis enzyme may besynthesized and subsequently ligated together. Preferably, theantithrombosis enzyme gene is synthesized as 10-20 separateoligonucleotides which are subsequently linked together. The individualoligonucleotides contain 5′ or 3′ overhangs for complementary assembly.

Following synthesis of the oligomers and cleavage of the desired vector,assembly of the antithrombosis enzyme gene may be achieved in one ormore steps by techniques well known in the art. Once assembled, the genewill be characterized by sequences which are recognized by restrictionendonucleases, including unique restriction sites for direct assemblyinto a cloning or an expression vector; preferential condons based uponthe host expression system to be used: and a sequence which, whentranscribed, produces a mRNA with minimal secondary structure. Properassembly maybe confirmed by nucleotide sequencing, restriction mapping,and expression of a biologically active antithrombosis enzyme in asuitable host.

It will be understood by those of skill in the art that, due to thedegeneracy of the genetic code, many different synthetic DNAs will becapable of encoding the antithrombosis enzyme of this invention. It willalso be apparent that many of these DNAs will be faithfully expressed inhost transformed with them. Therefore, the present invention relates tonot only one, but all DNA molecules which encode the desiredantithrombosis enzyme and which can be expressed by one or more hoststransformed with them. Most of these DNA molecules will be capable ofhybridizing to one another under moderately stringent conditions.

The DNA sequences and recombinant DNA molecules of the present inventionmay be inserted into and expressed using a wide variety of vectors. Assuch, the DNA sequence encoding the antithrombosis enzyme of theinvention must be operatively linked to an expression control sequence.The term “operatively linked”, as used herein refers to a positioning ina vector so that transcription and translation of the coding sequence isdirected by the control sequence. Useful vectors may consist of segmentsof chromosomal, non-chromosomal and synthetic DNA sequences, such asvarious known derivatives of SV40, bovine papilloma virus, adenovirusand cytomegalovirus and known bacterial plasmids, e.g., plasmids from E.coli including colEl, pCR1, pBR322, pMB9 and RP4; phage DNAs, e.g., thenumerous derivatives of lambda phage, e.g., NM 989, and other DNAphages, e.g., M13 and other Filamentous single-stranded DNA phages;vectors useful in yeasts, such as the 2 μm plasmid; vectors useful inanimal cells, such as those containing SV40 adenovirus andretrovirus-derived DNA sequences; commercially available high expressionvectors, e.g., the pGEM series and the lambda Zap vectors; and vectorsderived from combinations of plasmids and phage DNAs, such as plasmidswhich have been modified to employ phage DNA or other derivativesthereof.

Such expression vectors are also characterized by at least oneexpression control sequence. When the DNA sequences of this inventionare inserted in the vector they should be operatively linked to suchexpression control sequence in order to control and to regulate theexpression of that cloned DNA sequence. Examples of useful expressioncontrol sequences include the malE system, the OmpA system, the lacsystem, the trp system, the tac system, the trc system, major operatorand promoter regions of phage lambda, the control region of fd coatprotein, the glycolytic promoters of yeast, e.g., the promoter for3-phosphoglycerate kinase, the promoters of yeast acid phosphatase,e.g., Pho5, the promoters of the yeast-mating factors, and promotersderived from polyoma, adenovirus, retrovirus, and simian virus, e.g.,the early and late promoters of SV40, and other sequences known tocontrol the expression of genes of prokaryotic or eukaryotic cells andtheir viruses or combinations thereof.

Furthermore, within each specific expression vector, various sites maybe selected for insertion of DNA sequences encoding the antithrombosisenzyme or a fusion protein of this invention. These sites are usuallydesignated by the restriction endonuclease which cuts them. They arewell recognized by those of skill in the art. It is, of course, to beunderstood that an expression vector useful in this invention need nothave a restriction endonuclease site for insertion of the chosen DNAfragment. Instead, the vector may be joined to the fragment byalternative means.

The expression vector, and in particular the site chosen therein forinsertion of a selected DNA fragment and its operative linking thereinto an expression control sequence, is determined by a variety offactors, e.g., number of sites susceptible to a particular restrictionenzyme, size of the protein to expressed, susceptibility of the desiredprotein to proteolytic degradation by host cell enzymes, contaminationor binding of the protein to be expressed by host cell proteinsdifficult to remove during purification, expression characteristics,such as the location of start and stop codons relative to the vectorsequences, and other factors recognized by those of skill in the art.The choice of a vector and an insertion site for a DNA sequence isdetermined by a balance of these factors, not all selections beingequally effective for a given case.

Useful hosts which may be transformed with these vectors and which maybe employed to express the polypeptides of this invention may includewell known eukaryotic and prokaryotic hosts, such as strains of E. coli,such as E. coli SG-936, E. coli HB 101, E. coli W3110, E. coli X1776, E.coli X2282, E. coli DHI, and E. coli MRC1, Pseudomonas, Bacillus, suchas bacillus subtilis, Streptomyces, yeasts and other fungi, animalcells, such as COS cells and CHO cells, human cells, insect cells andplant cells in tissue culture.

Of course, not all host/expression vector combinations will functionwith equal efficiency in expressing the DNA sequences of this inventionor in producing the antithrombosis enzyme or fusion polypeptide.However, a particular selection of a host-expression vector combinationmay be made by those of skill in the art, after due consideration of theprinciples set forth herein without departing from the scope of thisinvention. For example, the selection should be based on a balancing ofa number of factors. These include, for example, compatibility of thehost and vector, toxicity of the proteins encoded by the DNA sequence tothe host, ease of recovery of the desired protein, expressioncharacteristics of the DNA sequences and the expression controlsequences operatively linked to them, biosafety, costs and the folding,form or any other necessary post-expression modifications of the desiredprotein.

Constructing a λ cDNA Library from the Venom Gland

After three days of venom extraction, Agkistrodon acutus snakes weresacrificed by decapitation. The venom gland was collected andimmediently frozen in liquid nitrogen. Total RNA was extracted from thevenom gland with isothiocyanate-phenol-chloride. mRNA was purified fromthe total RNA by an oligo(dT)-cellulose column. The first DNA strand wassynthesized by reverse transcriptase. The second DNA strand was thensynthesized and EcoRI adapters were ligated to the ends of the cDNA.This cDNA was ligated into λ gt11 vector and packaged. The packaged DNAwas titered and found to contain 1.1×10⁶ recombinants. Finally, 1.7×¹⁰pfu/ml were amplified to make the cDNA library.

Primers for cDNA Amplification of the Antithrombosis Polypeptides

One forward primer PF2 was designed according to the N-terminal aminoacid sequence of the A chain.

-   PF2: Y E G H C Y, 5′ TATGAAGGGCATTGCTACAA 3′

Another forward primer was designed from the 5′-terminal of the cDNAsequence of IX/X-Binding Protein containing the start codon (ATG):

-   PF3:5′ CCATGGGGCGATTCATCTTC 3′

A reverse primer (PR1) was designed from the 3′-terminal of the cDNAsequence of the IX/X-Binding Protein containing the stop codon (TGA).

-   PR1:5′ CAGCTGCATCTTCAGACTA 3′

The λ gt11 Forward Primer and Reverse Primer were also used to amplifythe λ cDNA.

λ gt11 Forward Primer: 5′ GGTGGCGACGACTCCTGGAGCCCG 3′ λ gt11 ReversePrimer: 5′ TTGACACCAGACCAACTGGTAATG 3′

Amplification of Clones Encoding the Antithrombosis Polypeptides

The λ cDNA library was used as the template for PCR amplification. ThePCR products were analyzed by 1.5% agarose gel electrophoresis andpurified from low temperature melting gel. The PCR products were,treated with Klenow fragment to generate blunt-ended DNA and insertedinto the pBluscript vector at the SmaI site with the T4 DNA ligase.Fresh competent E. coli was transformed with the ligation solution andpositive colonies were selected by color. The positive clones weredigested with HindIII/BamHI and sequenced with T3 and T7 primers and theT7 sequencing Kit (Promega).

One of the clones amplified with PF2/poly(T) primers, Clone A, contains544 bp as shown in SEQ ID NO: 1 which encodes the A chain of theantithrombosis enzyme from EGHCY to the carboxyl end.

One of the clone amplified with PF3/PR1 primers, Clone B, contains about500 bp as shown in FIG. 1 which putatively encodes the B chain of theantithrombosis enzyme.

The λ cDNA library was further screened with clone A as the probe.Several positive clones with inserts ranging from 500 bp to 1.2 kb havebeen selected to be sequenced.

Pharmaceutical Indications

Pathogenic platelet aggregation may yield thrombi (blood clots) whichocclude blood flow to dependent tissues and lead to a variety oflife-threatening vascular diseases, such as myocardial infarction,stroke, pulmonary embolism, deep vein thrombosis, peripheral arterialocclusion and other blood system thromboses. Methods of preventing orinhibiting platelet aggregation and release are desirable in thetreatment of these diseases. Inhibition of platelet aggregation may alsobe desirable in dialysis, storage of platelets in platelet concentrates,and following vascular surgery such as angioplasty.

The antithrombosis enzyme of this invention is effective for thetreatment of thrombotic diseases, including, but not limited to,angiopathic thrombosis, cerebral thrombosis, acute thrombosis,peripheral anginaphraxis, ischemic cerebral vascular disease, unstableangina, unstable stenocardia, hemiparalysis caused by cerebralthrombosis, myocardial infarction, stroke, pulmonary embolism, deep veinthrombosis and peripheral arterial occlusion; restenosis followingarterial injury or invasive cardiological procedures; acute or chronicatherosclerosis; edema and inflammation; abnormal cell regulatoryprocesses (e.g. secretion, shape changes, proliferation); cancer andmetastasis; and neurodegenerative diseases. It may also used during therecovery period of cerebral hemorrhage to reduce or inhibit blood clotin the brain.

Toxicology Studies

Acute Toxicity

The acute toxicity of the antithrombosis enzyme was studied with ratsand mice and two injection routes (i.t.v. and i.p.). The results areshown below:

TABLE 1 animal injection routes LD₅₀(95% reliability) mouse i.t.v. 14.1u/kg (9.7˜17.5 u/kg) i.p. 23.3 u/kg(18.4˜29.5 u/kg) rat i.t.v. 14.1u/kg(22.2˜17.2 u/kg) i.p. 14.1 u/kg(46.7˜63.5 u/kg)

Long-term Toxicity

For 8 weeks, rats were given the enzyme (i.t.v.) at dosagespharmaceutically equivalent to 12.5, 50, 200 times that's usedclinically for adult humans. Neither abnormal behavior nor toxic symptomwas observed. The consumption of food and water, net weight increase,various biochemical indexes and coefficients of major organs and tissuesshowed no visible change among the testing groups. Pathologicalhistology examination of major organs did not show distinct toxicosispathology changes.

Side Effects & Contra-indications

The enzyme has no neural toxicity and is non-hemorrhagic. No obviousside effects have been noticed in clinical use.

The Antithrombosis Enzyme Polypeptides, Antibodies and Hybridomas

A variety of methodologies known in the art can be utilized to obtainthe polypeptide of the present invention. The polypeptide may bepurified from a snake which naturally produces the polypeptide,chemically synthesized, or expressed by recombinant techniques.

Any eukaryotic organism can be used as a source for the polypeptide ofthe invention, as long as the source organism expresses such apolypeptide. One skilled in the art can readily follow known methods forisolating proteins in order to obtain the polypeptide free of naturalcontaminants. These include, but are not limited to: size-exclusionchromatography, HPLC, ion-exchange chromatography, and immuno-affinitychromatography.

The present invention relates to an antibody having binding affinity tothe antithrombosis enzyme polypeptide. The present invention alsorelates to an antibody having specific binding affinity to theantithrombosis enzyme polypeptide. Such an antibody may be isolated bycomparing its binding affinity to the antithrombosis enzyme polypeptidewith its binding affinity to another polypeptide. Those which bindselectively to the antithrombosis enzyme would be chosen for use inmethods requiring a distinction between the antithrombosis enzyme andother polypeptides.

The antithrombosis enzyme polypeptide of the present invention can beused in a variety of procedures and methods, such as for the generationof antibodies, for use in identifying pharmaceutical compositions, andfor studying DNA/protein interaction.

The antithrombosis enzyme polypeptide of the present invention can beused to produce antibodies or hybridomas. One skilled in the art willrecognize that if an antibody is desired, such a peptide would begenerated as described herein and used as an immunogen. The antibodiesof the present invention include monoclonal and polyclonal antibodies,as well fragments of these antibodies, and humanized forms. Humanizedforms of the antibodies of the present invention may be generated usingone of the procedures known in the art such as chimerization or CDRgrafting. The present invention also relates to a hybridoma whichproduces the above-described monoclonal antibody, or binding fragmentthereof. A hybridoma is an immortalized cell line which is capable ofsecreting a specific monoclonal antibody.

In general, techniques for preparing monoclonal antibodies andhybridomas are well known in the art (Campbell, Monoclonal AntibodyTechnology: Laboratory Techniques in Biochemistry and Molecular Biology,Elsevier Science Publishers, Amsterdam, The Netherlands (1984); St.Groth et al., J. Immunol. Methods 35:1-21(1980)). Any animal (mouse,rabbit, and the like) which is known to produce antibodies can beimmunized with the selected polypeptide. Methods for immunization arewell known in the art. Such methods include subcutaneous orintraperitoneal injection of the polypeptide. One skilled in the artwill recognize that the amount of polypeptide used for immunization willvary based on the animal which is immunized, the antigenicity of thepolypeptide and the site of injection.

The polypeptide may be modified or administered in an adjuvant in orderto increase the peptide antigenicity. Methods of increasing theantigenicity of a polypeptide are well known in the art. Such proceduresinclude coupling the antigen with a heterologous protein (such asglobulin or β-galactosidase) or through the inclusion of an adjuvantduring immunization.

For monoclonal antibodies, spleen cells from the immunized animals areremoved, fused with myeloma cells, such as SP2/0-Ag14 myeloma cells, andallowed to become monoclonal antibody producing hybridoma cells. Any oneof a number of methods well known in the art can be used to identify thehybridoma cell which produces an antibody with the desiredcharacteristics. These include screening the hybridomas with an ELISAassay, western blot analysis, or radioimmunoassay (Lutz et al., Exp.Cell Res. 175:109-124(1988)). Hybridomas secreting the desiredantibodies are cloned and the class and subclass is determined usingprocedures known in the art (Campbell, Monoclonal Antibody Technology:Laboratory Techniques in Biochemistry and Molecular Biology, supra(1984)).

For polyclonal antibodies, antibody containing antisera are isolatedfrom the immunized animal and screened for the presence of antibodieswith the desired specificity using one of the above-describedprocedures. The above-described antibodies may be detectably labeled.Antibodies can be detectably labeled through the use of radioisotopes,affinity labels (such as biotin, avidin, and the like), enzymatic labels(such as horse radish peroxidase, alkaline phosphatase, and the like)fluorescent labels (such as FITC or rhodamine, and the like),paramagnetic atoms, and the like. Procedures for accomplishing suchlabeling are well-known in the art, for example, see (Stemberger et al.,J. Histochem. Cytochem. 18:315(1970); Bayer et at., Meth. Enzym.62:308(1979); Engval et al., Immunot. 109:129(1972); Goding, J. Immunol.Meth. 13:215(1976)). The labeled antibodies of the present invention canbe used for in vitro, in vivo, and in situ assays to identify cells ortissues which express a specific peptide.

The above-described antibodies may also be immobilized on a solidsupport. Examples of such solid supports include Lay plastics such aspolycarbonate, complex carbohydrates such as agarose and sepharose,acrylic resins and such as polyacrylamide and latex beads. Techniquesfor coupling antibodies to such solid supports are well known in the art(Weir et al., Handbook of Experimental Immunology 4th Ed., BlackwellScientific Publications, Oxford, England, Chapter 10(1986); Jacoby etal., Meth. Enzym. 34 Academic Press, N.Y. (1974)). The immobilizedantibodies of the present invention can be used for in vitro, in vivo,and in situ assays as well as in immunochromotography.

Furthermore, one skilled in the art can readily adapt currentlyavailable procedures, as well as the techniques, methods and kitsdisclosed above with regard to antibodies, to generate peptides capableof binding to a specific peptide sequence in order to generaterationally designed antipeptide peptides, for example see Hurby et al.,Application of Synthetic Peptides: Antisense Peptides, In SyntheticPeptides, A User's Guide, W. H. Freeman, NY, pp. 289-307(1992), andKaspczak et al., Biochemistry 28:9230-8(1989).

The present invention encompasses a method of detecting theantithrombosis enzyme polypeptide in a sample, comprising: a) contactingthe sample with an above-described antibody, under conditions such thatimmunocomplexes form, and b) detecting the presence of said antibodybound to the polypeptide. In detail, the methods comprise incubating atest sample with one or more of the antibodies of the present inventionand assaying whether the antibody binds to the test sample.

Conditions for incubating an antibody with a test sample vary.Incubation conditions depend on the format employed in the assay, thedetection methods employed, and the type and nature of the antibody usedin the assay. One skilled in the art will recognize that any one of thecommonly available immunological assay formats (such asradioimmunoassays, enzyme-linked immunosorbent assays, diffusion basedOuchterlony, or rocket immunofluorescent assays) can readily be adaptedto employ the antibodies of the present invention. Examples of suchassays can be found in Chard, An Introduction to Radioimmunoassay andRelated Techniques Elsevier Science Publishers, Amsterdam, TheNetherlands (1986); Bullock et al., Techniques in Immunocytochemistry,Academic Press, Orlando, Fla. Vol. 1 (1982), Vol. 2 (1983), Vol. 3(1985); Tijssen, Practice and Theory of Enzyme Immunoassays: LaboratoryTechniques in Biochemistry and Molecular Biology, Elsevier SciencePublishers, Amsterdam, The Netherlands (1985).

The immunological assay test samples of the present invention includecells, protein or membrane extracts of cells, or biological fluids suchas blood, serum, plasma, or urine. The test sample used in theabove-described method will vary based on the assay format, nature ofthe detection method and the tissues, cells or extracts used as thesample to be assayed. Methods for preparing protein extracts or membraneextracts of cells are well known in the art and can be readily beadapted in order to obtain a sample which is capable with the systemutilized.

This invention relates to a kit containing all the necessary reagents tocarry out the previously described methods of detection. The kit maycomprise: i) a first container means containing an above-describedantibody, and ii) second container means containing a conjugatecomprising a binding partner of the antibody and a label. In anotherpreferred embodiment, the kit further comprises one or more othercontainers comprising one or more of the following: wash reagents andreagents capable of detecting the presence of bound antibodies.

Examples of detection reagents include, but are not limited to, labeledsecondary antibodies, or in the alternative, if the primary antibody islabeled, the chromophoric, enzymatic, or antibody binding reagents whichare capable of reacting with the labeled antibody. The compartmentalizedkit may be as described above for nucleic acid probe kits. One skilledin the art will readily recognize that the antibodies described in thepresent invention can readily be incorporated into one of theestablished kit formats which are well known in the art.

Pharmaceutical Formulations and Modes of Administration

The antithrombosis enzyme of the present invention may be formulatedinto pharmaceutically acceptable compositions for inhibiting boththrombin- and platelet mediated functions in a patient or inextracorporeal blood.

The dosage and dose rate of the antithrombosis enzyme of this inventionwill depend on a variety of factors, such as the patient's body weight,the specific pharmaceutical composition used, and the object of thetreatment, i.e., therapy or prophylaxis, the nature of the thromboticdisease to be treated, and the judgment of the treating physician. Apharmaceutically effective amount of the antithrombosis enzyme of thisinvention will normally be in the dosage range of between about0.001-500 mg/kg body weight, preferably about 0.1-50 mg/kg body weight.For the treatment of extracorporeal blood, the antithrombosis enzymeshould be used at about 0.005-50 μg/ml, preferably at about 0.5-5 μg/mlof extracorporeal blood. It should be understood that other dosagesoutside of these illustrative ranges may be employed in thepharmaceutical compositions of this invention.

The antithrombosis enzyme that affects the disorders of interest can beadministered in pharmaceutically acceptable formulations where it ismixed with suitable carriers or excipient(s). These formulations can beadministered by standard routes, including, but not limited to,intracerebroventricular, intracerebral, intravaginal, intrauterine orparenteral (e.g., intravenous, intraspinal, subcutaneous orintramuscular) route.

In treating a patient exhibiting a disorder of interest, atherapeutically effective amount of a agent or agents it; isadministered. A therapeutically effective dose refers to that amount ofthe compound that results in amelioration of symptoms or a prolongationof survival in a patient.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀.Compounds which exhibit large therapeutic indices are preferred. Thedata obtained from these cell culture assays and animal studies can beused in formulating a range of dosage for use in human. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized.

For any compound used in the method of the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. For example, a dose can be formulated in animal modelsto achieve a circulating plasma concentration range that includes theIC₅₀ as determined in cell culture (i.e., the concentration of the testcompound which achieves a half-maximal disruption of the proteincomplex, or a half-maximal is inhibition of the cellular level and/oractivity of a complex component). Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by HPLC.

The dosage of the antithrombosis enzyme will depend on the disease stateor condition being treated and other clinical factors such as weight andcondition of the patient and the route of administration of thecompound. It is to be understood that the present invention hasapplication for both human and veterinary use. The present inventioncontemplates single as well as multiple administrations, given eithersimultaneously or over an extended period of time.

The exact formulation, route of administration and dosage can be chosenby the individual physician in view of the patient's condition. (See e.Fingl et al., in The Pharmacoloaical Basis of Therapeutics, 1975, Ch. 1p. 1). It should be noted that the attending physician would know how toand when to terminate, interrupt, or adjust administration due totoxicity, or to organ dysfunctions. Conversely, the attending physicianwould also know to adjust treatment to higher levels if the clinicalresponse were not adequate (precluding toxicity). The magnitude of anadministrated dose in the management of the oncogenic disorder ofinterest will vary with the severity of the condition to be treated andto the route of administration. The severity of the condition may, forexample, be evaluated, in part, by standard prognostic evaluationmethods. Further, the dose and perhaps dose frequency, will also varyaccording to the age, body weight, and response of the individualpatient. A program comparable to that discussed above may be used inveterinary medicine.

Depending on the specific conditions being treated, such agents may beformulated and administered systemically or locally. Techniques forformulation and administration may be found in Remington'sPharmaceutical Sciences, 18th ed., Mack Publishing Co., Easton, Pa.(1990). Suitable routes may include intestinal administration orparenteral delivery, including intramuscular, subcutaneous,intramedullary injections, as well as intrathecal, directintraventricular, intravenous, intraperitoneal, intranasal, orintraocular injections, just to name a few.

For injection, the agents of the invention may be formulated in aqueoussolutions, preferably in physiologically compatible buffers such asHanks's solution, Ringer's solution, or physiological saline buffer. Forsuch transmucosal administration, penetrants appropriate to the barrierto be permeated are used in the formulation. Such penetrants aregenerally known in the art.

Use of pharmaceutically acceptable carriers to formulate the compoundsherein disclosed for the practice of the invention into dosages suitablefor systemic administration is within the scope of the invention. Withproper choice of carrier and suitable manufacturing practice, thecompositions of the present invention, in particular, those formulatedas solutions, may be administered parenterally, such as by intravenousinjection. The compounds can be formulated readily usingpharmaceutically acceptable carriers well known in the art into dosagessuitable for oral administration. Such carriers enable the compounds ofthe invention to be formulated as tablets, pills, capsules, liquids,gels, syrups, slurries, suspensions and the like, for oral ingestion bya patient to be treated.

Agents intended to be administered intracellularly may be administeredusing techniques well known to those of ordinary skill in the art. Forexample, such agents may be encapsulated into liposomes, thenadministered as described above. Liposomes are spherical lipid bilayerswith aqueous interiors. All molecules present in an aqueous solution atthe time of liposome formation are incorporated into the aqueousinterior. The liposomal contents are both protected from the externalmicroenvironment and, because liposomes fuse with cell membranes, areefficiently delivered into the cell cytoplasm. Additionally, due totheir hydrophobicity, small organic molecules may be directlyadministered intracellularly.

Pharmaceutical compositions suitable for use in the present inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve its intended purpose. Determination of theeffective amounts is well within the capability of those skilled in theart, especially in light of the detailed disclosure provided herein. Inaddition to the active ingredients, these pharmaceutical compositionsmay contain suitable pharmaceutically acceptable carriers comprisingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations which can be used pharmaceutically. Thepreparations formulated for oral administration may be in the form oftablets, dragees, capsules, or solutions. The pharmaceuticalcompositions of the present invention may be manufactured in a mannerthat is itself known, e.g., by means of conventional mixing, dissolving,granulating, dragee-making, levitating, emulsifying, encapsulating,entrapping or lyophilizing processes.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.

Dosage & Parental Administration

The antithrombosis enzyme could be administered to patients byintravenous injection. The enzyme is supplied as sterile lyophilizedwhite powder in vials. Each vial contains 0.25 unit. For treatment,dissolve this drug aseptically by adding proper sterile water. Thesolution is diluted with 250 ml of 0.9% Sodium Chloride and is givenintravenously at the rate of 45 drops/min. 0.25 unit is given per day.

For acute treatment 0.5 u is given on the first day and 0.25 u/dayafterwards. The course of treatment is two weeks.

The drug should be administered under the supervision of a physician.

Perform intradermal test before administration. The drug is diluted into0.0025 u/ml. Take 0.1 ml and inject intradermally. Observe the resultafter 20 min. The drug should not be given to the patient if the testresult is positive.

Platelet count should be checked in regular intervals (once a week). Ifthe platelet count falls below 80,000/mm³, the treatment should bediscontinued immediately.

Gene Therapy with the ATE

The antithrombosis enzyme or its genetic sequences will be useful ingene therapy (reviewed in Miller, Nature 357:455-460, 1992). Millerstates that advances have resulted in practical approaches to human genetherapy that have demonstrated positive initial results. An in vivomodel of gene therapy for human severe combined immunodeficiency isdescribed in Ferrari, et al., Science 251:1363-1366, (1991). The basicscience of gene therapy is described in Mulligan, Science 260:926-931,(1993).

In one preferred embodiment, one or more expression vector containingthe ATE coding sequence is inserted into cells, the cells are grown invitro and then infused in large numbers into patients.

The gene therapy may involve the use of an adenovirus containing ATEcDNA targeted to a vascular tissue, systemic ATE increase byimplantation of engineered cells, or injection of naked ATE DNA intoappropriate tissues.

Expression vectors derived from viruses such as retroviruses, vacciniavirus, adenovirus, adeno-associated virus, herpes viruses, several RNAviruses, or bovine papilloma virus, may be used for delivery ofnucleotide sequences (e.g., cDNA) encoding recombinant ATE polypeptidesinto the targeted cell population (e.g., tumor cells). Methods which arewell known to those skilled in the art can be used to constructrecombinant viral vectors containing coding sequences. See, for example,the techniques described in Maniatis et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, N.Y. (1989), and inAusubel et al., Current Protocols in Molecular Biology, GreenePublishing Associates and Wiley Interscience, N.Y. (1989).Alternatively, recombinant nucleic acid molecules encoding proteinsequences can be used as naked DNA or in reconstituted system e.g.,liposomes or other lipid systems for delivery to target cells (See e.g.,Felgner et al., Nature 337:387-8, 1989). Several other methods for thedirect transfer of plasmid DNA into cells exist for use in human genetherapy and involve targeting the DNA to receptors on cells bycomplexing the plasmid DNA to proteins. See, Miller, supra.

In its simplest form, gene transfer can be performed by simply injectingminute amounts of DNA into the nucleus of a cell, through a process ofmicroinjection. Capecchi MR, Cell 22:479-88 (1980). Once recombinantgenes are introduced into a cell, they can be recognized by the cell'snormal mechanisms for transcription and translation, and a gene productwill be expressed. Other methods have also been attempted forintroducing DNA into larger numbers of cells. These methods include:transfection, wherein DNA is precipitated with CaPO₄ and taken intocells by pinocytosis (Chen C. and Okayama H, Mol. Cell Biol. 7:2745-52(1987)); electroporation, wherein cells are exposed to large voltagepulses to introduce holes into the membrane (Chu G. et al., NucleicAcids Res., 15:1311-26 (1987)); lipofection/liposome fusion, wherein DNAis packaged into lipophilic vesicles which fuse with a target cell(Felgner PL., ( et al., Proc. Natl. Acad. Sci. USA. 84:7413-7 (1987));and particle bombardment using DNA bound to small projectiles (Yang NS.et al., Proc. Natl. Acad. Sci. 87:9568-72 (1990)). Another method forintroducing DNA into cells is to couple the DNA to chemically modifiedproteins.

It has also been shown that adenovirus proteins are capable ofdestabilizing endosomes and enhancing the uptake of DNA into cells. Theadmixture of adenovirus to solutions containing DNA complexes, or thebinding of DNA to polylysine covalently attached to adenovirus usingprotein crosslinking agents substantially improves the uptake andexpression of the recombinant gene. Curiel DT et al., Am. J. Respir.Cell. Mol. Biol., 6:247-52 (1992).

As used herein “gene transfer” means the process of introducing aforeign nucleic acid molecule into a cell. Gene transfer is commonlyperformed to enable the expression of a particular product encoded bythe gene. The product may include a protein, polypeptide, anti-sense DNAor RNA, or enzymatically active RNA. Gene transfer can be performed incultured cells or by direct administration into animals. Generally genetransfer involves the process of nucleic acid contact with a target cellby non-specific or receptor mediated interactions, uptake of nucleicacid into the cell through the membrane or by endocytosis, and releaseof nucleic acid into the cytoplasm from the plasma membrane or endosome.Expression may require, in addition, movement of the nucleic acid intothe nucleus of the cell and binding to appropriate nuclear factors fortranscription.

As used herein “gene therapy” is a form of gene transfer and is includedwithin the definition of gene transfer as used herein and specificallyrefers to gene transfer to express a therapeutic product from a cell invivo or in vitro. Gene transfer can be performed ex vivo on cells whichare then transplanted into a patient, or can be performed by directadministration of the nucleic acid or nucleic acid-protein complex intothe patient.

In another preferred embodiment, a vector having nucleic acid sequencesencoding ATE is provided in which the nucleic acid sequence is expressedonly in specific tissue. Methods of achieving tissue-specific geneexpression as set forth in International Publication No. WO 93/09236,filed Nov. 3, 1992 and published May 13, 1993.

In all of the preceding vectors set forth above, a further aspect of theinvention is that the nucleic acid sequence contained in the vector mayinclude additions, deletions or modifications to some or all of thesequence of the nucleic acid, as defined above.

In another preferred embodiment, a method of gene replacement is setforth. “Gene replacement” as used herein means supplying a nucleic acidsequence which is capable of being expressed in vivo in an animal andthereby providing or augmenting the function of an endogenous gene whichis missing or defective in the animal.

All publications referenced are incorporated by reference herein,including the nucleic acid sequences and amino acid sequences listed ineach publication.

Other embodiments of this invention are disclosed in the followingclaims.

1. A method for dissolving a thrombus in a mammal, comprising the stepof administering to said mammal a pharmaceutically effective amount ofan isolated, purified or recombinant antithrombosis enzyme having thefollowing characteristics: the molecular weight of said enzyme isbetween about 28 kD and about 32 kD when analyzed by polyacrylamide gelelectrophoresis, the aspartic acid content of said enzyme is betweenabout 2% and about 5%, the glutamic acid content of said enzyme isbetween about 2% and about 5% said enzyme hydrolyzes fibrin, dissolvesblood clots, and Prevents platelet aggregation.
 2. A method for treatinga thrombosis related disease in a mammal, comprising the step ofadministering to said mammal a pharmaceutically effective amount of anenzyme of claim
 1. 3. The method of claim 1, wherein said thrombosisrelated disease is selected from the group consisting of myocardialinfaction, restenosis, unstable angina and cerebral thrombosis.
 4. Amethod for inhibiting human platelet aggregation induced by a fibrinagonist selected from the group consisting of ADP, epinephrine andthrombin, comprising the step of administering a pharmaceuticallyeffective amount of an enzyme of claim
 1. 5. The method of claim 1,wherein said enzyme has no detectable hydrolysis effect on casein. 6.The method of claim 1, wherein said enzyme comprises Ca⁺⁺.
 7. The methodof claim 1, wherein the amino terminus of said enzyme is aspartic acid.8. The method of claim 1, wherein said enzyme comprises two polypeptidechains of about 14 kD to about 16 kD when analyzed by polyacrylamide gelelectrophoresis.
 9. The method of claim 8, wherein one of said twopolypeptide chains comprises an amino acid sequence, from left to rightin the direction from the amino terminus to the carboxy terminus,represented by the formula, SEQ ID NO: 3:Asp-Cys-Ser-Ser-Asp-Trp-Ser-Ser-Tyr-Glu-Gly-His-Cys-Tyr-Lys-Val-Phe-Lvs-Gln-Ser-Lys-Thr-Trp-Thr-Asp-Ala-Glu-Ser-Phe-.10. The method of claim 8, wherein one of said two polypeptide chainscomprises an amino sequence, from left to right in the direction fromthe amino terminus to the carboxy terminus, represented by the formula,SEQ ID NO:4:Asp-Cys-Pro-Ser-Glu-Trp-Ser-Ser-Tyr-Glu-Gly-Phe-Cys-Tyr-Lvs-Pro-Phe-.11. The method of claim 8, wherein said one of two polypeptide chainscomprises an amino acid sequence of SEQ ID NO:
 2. 12. The method ofclaim 1, wherein said enzyme is crystallized.